A bacterial arginine-agmatine exchange transporter involved in extreme acid resistance

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.     

Outer and inner membrane proteins compose an arginine-agmatine exchange system in Chlamydophila pneumoniae

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts L-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of L-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of L-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, L-lysine and L-ornithine, which inhibit the E. coli AdiC antiporter.     

Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli

The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine alpha-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.     

Protonation of glutamate 208 induces the release of agmatine in an outward-facing conformation of an arginine/agmatine antiporter

Virulent enteric pathogens have developed several systems that maintain intracellular pH to survive extreme acidic conditions. One such mechanism is the exchange of arginine (Arg(+)) from the extracellular region with its intracellular decarboxylated form, agmatine (Agm(2+)). The net result of this process is the export of a virtual proton from the cytoplasm per antiport cycle. Crystal structures of the arginine/agmatine antiporter from Escherichia coli, AdiC, have been recently resolved in both the apo and Arg(+)-bound outward-facing conformations, which permit us to assess for the first time the time-resolved mechanisms of interactions that enable the specific antiporter functionality of AdiC. Using data from ～1 μs of molecular dynamics simulations, we show that the protonation of Glu-208 selectively causes the dissociation and release of Agm(2+), but not Arg(+), to the cell exterior. The impact of Glu-208 protonation is transmitted to the substrate binding pocket via the reorientation of Ile-205 carbonyl group at the irregular portion of transmembrane (TM) helix 6. This effect, which takes place only in the subunits where Agm(2+) is released, invites attention to the functional role of the unwound portion of TM helices (TM6 Trp-202-Glu-208 in AdiC) in facilitating substrate translocation, reminiscent of the behavior observed in structurally similar Na(+)-coupled transporters.     

Sided functions of an arginine-agmatine antiporter oriented in liposomes

The arginine-dependent extreme acid resistance system helps enteric bacteria survive the harsh gastric environment. At the center of this multiprotein system is an arginine-agmatine antiporter, AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product, agmatine, resulting in a net extrusion of one "virtual proton" in each turnover. The random orientation of AdiC in reconstituted liposomes throws up an obstacle to quantifying its transport mechanism. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits substrate recognition and pH-dependent activity similar to those of the wild-type protein but loses function completely upon reaction with thiol reagents. The membrane-impermeant MTSES reagent can then be used as a cleanly sided inhibitor to silence those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. This approach allows steady-state kinetic analysis of AdiC in a sided fashion. Arginine and agmatine have similar Michaelis-Menten parameters for both sides of the protein, while the extracellular side selects arginine over argininamide, a mimic of the carboxylate-protonated form of arginine, more effectively than does the cytoplasmic side. Moreover, the two sides of AdiC have different pH sensitivities. AdiC activity increases to a plateau at pH 4 as the extracellular side is acidified, while the cytoplasmic side shows an optimal pH of 5.5, with further acidification inhibiting transport. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.     

Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily

The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded two-dimensional crystals of AdiC-W293L diffracting beyond 6 angstroms resolution from which we determined the projection structure at 6.5 angstroms resolution. The projection map showed 10-12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning alpha-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.     

Glutamate decarboxylase‐dependent acid resistance in orally acquired bacteria: function,distribution and biomedical implications of the gadBC operon

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate‐dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP‐dependent enzyme which performs a proton‐consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamate_in/γ‐aminobutyrate_out (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH‐dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non‐pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co‐regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA‐enriched foods possess health‐promoting properties.     

Isolation of Conditionally Putrescine-Deficient Mutants of Escherichia coli

Mutants defective in the conversion of arginine to putrescine were found by screening clones from mutagenized cultures for inability to produce urea during growth in arginine-supplemented media. Two partially blocked mutants were isolated; one was deficient in arginine decarboxylase and the other was deficient in agmatine ureohydrolase. As predicted from the pattern of putrescine synthesis in Escherichia coli, these mutants were conditionally putrescine-deficient. When grown in either minimal or ornithine-supplemented media, conditions which lead to preferential utilization of the ornithine to putrescine pathway, the mutants had normal intracellular polyamine levels. However, when the mutants were placed in arginine-supplemented media, the level of intracellular putrescine was lowered markedly. Under conditions where intracellular putrescine was 1% of normal, the doubling time of the mutants was increased approximately 10%. The putrescine-deficient mutants had wild-type morphology, normal levels of protein and ribonucleic acid (RNA), and stringent amino acid control of RNA synthesis.     

Characterization of an acid-dependent arginine decarboxylase enzyme from Chlamydophila pneumoniae

Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (alphabeta)(3) complex. The mature enzyme specifically catalyzed the decarboxylation of L-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5'-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.     

Uptake of Agmatine into Rat Brain Synaptosomes: Possible Role of Cation Channels

Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy- and temperature-dependent and saturable with a K _m of 18.83 ± 3.31 m M and a V _max of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na⁺,K⁺-ATPase inhibitor) or removal of extracellular Na⁺ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion-channel agents on agmatine uptake, only Ca²⁺-channel blockers inhibited the uptake, whereas a reduction in extracellular Ca²⁺ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na⁺-independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.     

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.     

Characterization of EvgAS-YdeO-GadE branched regulatory circuit governing glutamate-dependent acid resistance in Escherichia coli

Escherichia coli prefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the four E. coli systems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, gamma-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncovered evgAS and ydeO as potential regulators of gadE, now known to encode the essential activator of the gadA and gadBC genes. Examination of evgA and ydeO under normal expression conditions revealed that their products do activate gadE expression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary for gadE expression in stationary-phase cells grown in complex medium. The response regulator EvgA activates gadE directly and indirectly via induction of the AraC-like regulator ydeO. Evidence obtained using gadE-lacZ operon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream of gadE, indicating they all act directly at the gadE promoter. Since GadE controls the expression of numerous genes besides gadA and gadBC, the relevance of these regulatory circuits extends beyond acid resistance.     

Rapid Methods for Determining Decarboxylase Activity: Arginine Decarboxylase

A rapid biochemical method for the determination of arginine decarboxylase (EC 4.1.1.19) activity has been developed for use in the routine clinical microbiology laboratory and correlated with similar procedures for ornithine and lysine decarboxylase (EC 4.1.1.18) systems. It is based on the detection of agmatine, the amine end product formed during growth on a synthetic medium containing arginine as the key amino acid. A modified diacetyl reagent is used to detect this amine after a differential butanol extraction of the cultures. This procedure can be used to detect this amine after a 1- to 4-hr incubation period (with the use of an initial concentrated inoculum) or with an overnight culture. Thus, both an indirect measurement based on the alkalinization of the medium and a lengthy incubation period were avoided. Parameters for optimal enzyme activity and the pertinent enzyme systems involved in arginine and agmatine catabolism are discussed in detail.     

Control of Acid Resistance in Escherichia coli

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.     

Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.